Insulin-like growth factor-1 (IGF-1) stimulates the IGF binding protein system in rat theca interstitial cells

There has been considerable interest in rat ovarian insulin-like growth factor binding proteins IGFBPs because they are potent inhibitors of FSH action.In situ, IGFBP-2 and -4 and IGFBP-3 mRNAs are expressed in rat theca interstitial (TIC) and theca lutein cells respectively. Although much is known about IGFBPs in rat TIC at the mRNA level, the synthesis and regulation of IGFBP proteins remain poorly understood. The purpose of this study was to identify the species of IGFBPs produced by TIC and to determine the effects of LH and IGF-1 on their expression. This was accomplished by culturing rat TIC for 2 days in serum-free medium with graded doses of LH and/or IGF-I, and measuring IGFBP mRNAs in the cells and IGFBP proteins in the conditioned media by RT-PCR and Western immunoblotting respectively. The RT PCR analysis identified strong bands for IGFBP-2 and -4 mRNAs in TIC. In some treatments, the mRNAs for IGFBP-3 and -6 were also identified, but transcripts for IGFBP-1 and -5 were undetectable. Two species of IGFBPs were detected in the conditioned media of control (untreated) TIC, the 31 kDa IGFBP-2 and the 24 kDa (non-glycosylated) and 28 kDa (glycosylated) forms of IGFBP-4. There was no detectable IGFBP-5 and barely detectable amounts of IGFBP-3 and -6 in the conditioned media. Treatment with LH (0.2-20 μU/ml) caused no significant changes in the levels of the 31 kDa IGFBP-2 and the 24 kDa and 28 kDa IGFBP-4 bands, and there was no detectable IGFBP protease activity. In contrast, IGF-I (100 ng/ml) stimulated the expression of IGFBP-2, IGFBP-4 and a 17.5 kDa IGFBP-4 fragment. The immunoreactive IGFBP-4 fragment suggests the media contained an IGFBP-4 protease. The IGF-I effects were dose dependent (ED(50)=12.4±3.3 ng/ml). Co-treating TIC with LH (0.2-20 μU/ml) caused no significant change in the activity of IGF-I in stimulating the expression of IGFBP-2, IGFBP-4 and IGFBP-4 protease. We have demonstrated that IGF-I acts directly on rat TIC to stimulate the expression of the intrinsic IGFBP system. LH, either alone or together with IGF-I, did not significantly change the expression of TIC IGFBP proteins. Therefore, we hypothesize that IGF-I, but not LH, may be a physiologically important regulator of the IGFBP system in rat TIC. Because IGF-I is a potent stimulator of theca function, changes in the expression of this intrinsic IGFBP system could have new implications for ovarian androgen production, both at the physiologic and pathophysiologic levels.